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Introduction |
Choline, a major constituent of eukaryotic membrane
lipids, was previously thought to be an unusual structural feature of prokaryotes. Choline, in the form of choline phosphate or phosphorylcholine (ChoP),1 is found on
the teichoic acid of Streptococcus pneumoniae and has recently
been identified as a unique feature of LPS of Haemophilus influenzae (1). An mAb, TEPC-15, that specifically recognizes the ChoP structure has been used to show that the
ChoP epitope is also expressed on pili of pathogenic Neisseriae and a protein of unknown function in Pseudomonas
aeruginosa (reference 4, and Weiser, J.N., J. Goldberg, N. Pan, L. Wilson, and M. Virji, manuscript submitted for
publication).
In the case of H. influenzae, choline is acquired from the
environment and linked as ChoP to a hexose on the outer
core region of the LPS (1, 3, 5). Since the LPS of H. influenzae lacks the multiple O-linked saccharide units characteristic of the enterobacteriaceae, ChoP is located on the
cell surface. There is both inter- and intrastrain variation in
structure of the LPS as a result of differences in the composition and linkage of saccharides in the outer core (6). An
additional source of heterogeneity of the LPS is phase variation in the decoration of the LPS with the ChoP epitope.
The expression of the ChoP epitope on the H. influenzae
glycolipid requires the four genes of the lic1 locus (9). This
locus is present in all strains in a representative survey of
encapsulated and nontypable H. influenzae isolates, but is
not required for normal growth in vitro (10). The first gene
in lic1, licA, has homology to eukaryotic choline kinases. This gene contains an unusual feature consisting of variable
numbers of tandem repeats of the sequence 5
-(CAAT)-3
within the open reading frame (11). Variation in the number of repeat units by slipped-strand mispairing alters the
alignment of initiation codons with the licA open reading
frame creating a translational switch that results in spontaneous phase variation in expression of the ChoP epitope.
The frequency of on-off switching in the expression of the
ChoP epitope is ~10
2-3/generation, but varies from
strain to strain depending on the length of the repetitive sequence (1).
A gene with similarity to licA has also been noted in S. pneumoniae and in various mycoplasma species, including
Mycoplasma fermentans and pneumoniae (12, 13). The presence of the ChoP epitope on the cell surface of S. pneumoniae, H. influenzae, N. meningiditis, and M. pneumoniae, all
major pathogens residing in the human respiratory tract,
suggests that this structure may contribute to the ability of
these species to occupy their niche on this mucosal surface.
In S. pneumoniae, H. influenzae, N. meningitidis, N. gonorrhoeae, and P. aeruginosa there is also phase variation in the
expression of the ChoP epitope (1, 14). Since these pathogens also commonly cause invasive infection, we addressed whether ChoP may contribute to the ability of organisms
to reside in the human nasopharynx as well as their ability
to survive in the bloodstream by evasion of humoral immunity. As a model system we have selected H. influenzae;
it has already been documented that ChoP epitope expressing variants of a type b H. influenzae strain are more sensitive to the bactericidal effect of human serum than variants
lacking this structure (1). It was postulated that the difference in serum sensitivity was a result of naturally acquired
antibody against ChoP since the serum in this study had higher immunoglobulin G titers to LPS with the ChoP
epitope compared to LPS without the ChoP epitope.
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Materials and Methods |
Bacterial Strains, Media, and Chemicals.
H. influenzae strains
used for this study included H233, a nontypable clinical isolate
(Strain A860516) obtained from the collection of Dr. Loek van
Alphen (University of Amsterdam, The Netherlands). A kanamycin-resistant encapsulated type b strain (Eagan) and a mutant of
this strain with a deletion/insertion spanning the four genes in lic1
were used in animal experiments (15, 16). Type b strain RM7004
was used for structural analysis (9). H. influenzae strains were
grown in brain heart infusion broth supplemented with 1.5%
fildes enrichment with or without 1% agar (Difco Laboratories,
Detroit, MI). When specified, a chemically defined medium was
used with laboratory strain Rd, for which this medium is suitable
(17). Chemicals were purchased from Sigma Chemical Co. (St.
Louis, MO) unless otherwise specified.
Structural Analysis of LPS.
LPS from TEPC-15-reactive and
nonreactive colonies of strain RM7004 were isolated from BHI
broth-grown cells by the hot phenol-water extraction procedure
(8). Purified LPS were analyzed directly for sugar composition by
complete acid hydrolysis and gas liquid chromatography of the
derived acetylated reduced aldoses (8). O-deacylated LPS were
prepared by mild hydrazine treatment for comparative analysis by
electrospray ionization-mass spectrometry (ESI-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy (18). ESI-MS was
performed in the negative ion mode on a VG Quattro Mass
Spectrometer (Micromass, Manchester, UK) by direct infusion of
samples in 25% aqueous acetonitrile containing 0.5% acetic acid.
1H NMR spectra were recorded at 500 MHz on samples in D2O
at 37°C on a Bruker AMX 500 spectrometer with acetone (methyl
resonance: 
2.225 ppm) as the internal chemical shift standard.
Colony Immunoblotting.
Colonies lifted onto nitrocellulose
were immunoblotted to separate phase variants as previously described (1). The ChoP epitope on colonies lifted onto nitrocellulose was detected using a 1:10,000 dilution of mAbs against
ChoP, TEPC-15, or HAS (Statens SerumInstitut, Copenhagen,
Denmark), followed by alkaline phosphatase-conjugated anti-
mouse IgA or IgM, respectively. In samples obtained from nasal
washes, 20-200 colonies from each pup were immunoblotted to
determine the proportion of ChoP+ and colonies. After determining the phenotype with respect to ChoP expression, a single
colony was used to inoculate 5-ml BHI broth cultures for extraction of chromosomal DNA for use as a template for the PCR by
a published method (19).
Western Blot Transfer and Immunoblotting.
Electrotransfer of
proteins separated on 12% SDS-PAGE onto Immobilon-P (Millipore Co., Bedford, MA) and Western blotting were carried out as
previously described (1). Depletion of C-reactive protein (CRP)
and/or IgG from the pooled normal human serum (NHS) was
confirmed by Western blot analysis using an mAb to human CRP and polyclonal serum against human IgG, respectively.
Serum Bactericidal Assays.
Complement-mediated serum bactericidal activity was determined in NHS pooled from six random
adult donors and stored at 
80°C. Assays were performed with
20 µl of a suspension of midlog phase organisms (OD620 0.3-0.4)
diluted to 105 CFU/ml with HBSS (GIBCO BRL, Gaithersburg,
MD), 60 µl HBSS, 100 µl PBS, and 20 µl pooled NHS. After
incubation for 60 min at 37°C with rotation, the assay was
stopped by cooling to 4°C and dilutions were made for quantitative culture. To calculate the percentage of survival, colony
counts were compared to controls in which complement activity
had been eliminated by prior heating of the NHS to 56°C for 30 min. IgG was removed from NHS using a protein G column according to the manufacturer's instructions (Pharmacia Biotech
AB, Uppsala, Sweden). CRP was removed from the NHS by incubation at 4°C for 30 min with an equal volume of ChoP-coupled agarose beads (Pierce Chemical Co., Rockford, IL) that had
been washed in 0.02 M Tris (pH 7.2), 0.15 M NaCl, and 10 mM
CaCl2. Purified human CRP was dialyzed in PBS to remove sodium azide and its concentration was determined using the Micro
BCA protein assay (Pierce Chemical Co.). The purity of the human CRP was confirmed by SDS-PAGE. The requirement for
Ca2+ in CRP-mediated serum killing was tested by preincubation
of cells in CRP (5 µg/ml) for 15 min at 37°C in either the buffer described above or the same buffer in which 2 mM EDTA was
substituted for the 10 mM CaCl2. After washing the cells in
HBSS to remove unbound CRP, the cells were used in a bactericidal assay using CRP-depleted NHS as a source of complement
activity.
Binding of CRP to H. influenzae Variants.
ChoP+ and variants were grown to OD620 = 0.4, washed in PBS, and resuspended in 0.02 M Tris (pH 7.2), 0.15 M NaCl, and either 10 mM CaCl2 or 2 mM EDTA. Purified human CRP (1.0 µg/108
cells) was allowed to bind to the cells in the presence of 2.5% CRP-depleted NHS for additional buffering and to block nonspecific binding. After 15 min at 37°C with slow rotation, the
cells were washed in PBS and an aliquot was removed and treated
at 100°C in gel loading buffer for separation in 12% SDS-PAGE
and Western blot analysis. CRP was detected using an mAb to
human CRP followed by an alkaline phosphatase-conjugated
anti-mouse immunoglobulin.
Choline Incorporation.
H. influenzae was radiolabeled by adding [3H]choline (New England Nuclear Co., Boston, MA) to the
chemically defined medium (final concentration: 0.25 µCi/ml).
H. influenzae was grown to an OD620 of 0.3 and washed three
times in an equal volume of PBS. Aliquots were removed for colony immunoblotting and for determination of total cellular protein. The remainder of the sample was used to determine the incorporation of the label in whole cells.
Infant Rat Model of Nasopharyngeal Colonization.
Synchronized
pregnant Sprague-Dawley rats were purchased from Taconic
Farms (Germantown, N.Y.). 5-d-old infant rats were randomized
among litters. For intranasal inoculations, 10 µl of PBS-washed
midlog phase organisms adjusted to a density of 108 CFU/ml
were inoculated into the left anterior naris. Animals receiving different phenotypic variants were housed in separate cages. Colony
counts were performed to ensure the inocula were of the desired
density. The nasopharynx was cultured by the slow instillation of
20-40 µl of sterile PBS into the left naris and withdrawal of the
initial 10 µl discharge from the right naris. This ensured that the
fluid had passed through the nasopharynx. To assess the quantity
of organisms in nasal washes, serial dilutions in PBS were plated
on supplement brain heart infusion agar containing kanamycin
(20 µg/ml) to diminish the growth of non-Haemophilus contaminants.
Genotypic analysis of H. influenzae in human respiratory tract secretions.
Human respiratory tract secretions obtained from expectorated sputum or bronchoscopy were stored at 80°C and were
included only if shown subsequently to have a predominant
growth of H. influenzae on culture. Specimens containing purulent sputum were from nonbacteremic adult patients hospitalized
with pneumonia. The diagnosis of H. influenzae pneumonia was
based on microscopic examination of gram-stained sputum that
revealed >20:1 ratio of inflammatory to epithelial cells and profuse gram-negative coccobacilli with only rare or no other organisms (20). DNA was extracted from 50 µl of homogenized specimen by the addition of 1 µl of 1 M Tris-HCl (pH 7.6) and 1 µl
of proteinase K (5 µg/ml) followed by incubation at 35°C for 60 min and then for 20 min at 100°C. This DNA was used as a template to amplify the 5 region of lic1 using Taq polymerase and
primers 5-TCGAATCCGCAAAAGTCACCATTTATTGTGAAG-3 (forward) and 5-TGGAATTCGTCCGCCTAATATGCCAGATAAC-3 (reverse) by PCR. Conditions for PCR included thirty cycles in 1-2 mM Mg2+, denaturing at 94°C for 40 s,
annealing at 50°C for 40 s and extension at 60°C for 90 s. The
number of CAAT repeats was determined on the gel-extracted
PCR product by sequencing across this region using primer
5-TATTACATAATCTTTCAGCT-3.
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Results |
Structural Analysis of Variants in Expression of the ChoP
Epitope.
Comparative analysis of LPS from TEPC-15-reactive and -nonreactive colonies confirmed that the presence
of ChoP on the outer core region of the LPS accounted for
the difference. This was achieved by ESI-MS and 1H
NMR analysis of samples of O-deacylated LPS from a type
b strain. A notable feature of the 1H NMR that was particularly informative is the intense singlet at ca 3.25 ppm due
to the ChoP methyl protons (Fig. 1); a signal of significant
intensity is observed only in the O-deacylated LPS containing the ChoP epitope. It is now well established that H. influenzae express a heterogeneous mixture of LPS molecules. A structural model has been advanced, comprised of a conserved heptose-containing inner core trisaccharide moiety
attached via phosphorylated 3-deoxy-D-manno-octulosonic
acid to a lipid A component in which each of the heptose
residues can provide a point for elongation by hexose containing oligosaccharide chains or for attachment of noncarbohydrate substituents (3, 8). ESI-MS of O-deacylated LPS
from TEPC-15-nonreactive colonies revealed a series of
related structures differing in the number of hexose residues
(Table 1) in which populations of LPS glycoforms containing three to nine hexose residues were identified. An additional parallel series of ions corresponding to subpopulations of glycoforms containing ChoP substituents was
detected only for O-deacylated LPS from TEPC-15-reactive colonies. In a lic2 mutant of this type b strain, detailed
structural studies have established the molecular environment in which the ChoP structure is expressed (5).
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Fig. 1.
1H NMR spectra of O-deacylated LPS from TEPC-15-reactive (a) and -nonreactive (b) colonies of H. influenzae strain RM7004. The strong signal at ca 3.35 ppm in a is indicative of the presence of
ChoP. This signal is present only to the extent of ca 1% in b. Spectra were
recorded at 500 MHz in D2O containing 2 mM perdeutero EDTA and
10 mg/ml perdeutero SDS.
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Table 1
Negative Ion ESI-MS Data and Proposed Compositions for O-deacylated LPS from TEPC-15-nonreactive and -reactive Colonies of Type b H. influenzae (RM 7004)
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Contribution of ChoP to Susceptibility to Serum Bactericidal
Activity.
Phase variants of a nontypable clinical isolate differing in the expression of ChoP were compared for their
ability to survive the bactericidal effect of human serum.
Survival of the ChoP+ variant in 10% NHS for 60 min was
<1% compared to the ChoP variant of the same strain
(Fig. 2 A). The difference in serum sensitivity did not appear to be caused by the presence of naturally acquired antibody against ChoP since removal of IgG had no effect on
the increased susceptibility of the ChoP+ variant. However, killing of both H. influenzae variants appeared to be
dependent on the classical rather than alternative pathway of complement activation as the addition of 0.05 M Mg2+
EGTA to chelate divalent cations completely eliminated
serum killing of both variants (21).
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Fig. 2.
Serum bactericidal assay showing the contribution of immunoglobulin G and CRP to the survival of phase variants in the expression of ChoP in NHS. ChoP+ (solid bar) or ChoP (hatched bar) variants of
nontypable strain H233 were grown to midlog phase and treated for 60 min in 10% pooled NHS. The percentage of survival is the number of
CFUs remaining compared to controls in which complement activity was
inactivated. In A, determinations were carried out in the presence of untreated NHS (lane 1), NHS depleted of IgG (lane 2), NHS depleted of
IgG with the addition of 0.05 M Mg2+ EGTA (lane 3), NHS depleted of
IgG and CRP (lane 4), NHS depleted of CRP alone (lane 5), or NHS
depleted of CRP with the addition of purified human CRP (5 µg/ml)
(lane 6). In B, before use in the bactericidal assay with CRP-depleted
NHS, phase variants were pretreated with purified human CRP in the
presence of EDTA (lane 7) or Ca2+ (lane 8). Values are the geometric
mean of at least three determinations in duplicate ± SD.
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The Effect of CRP on Serum Killing of H. influenzae.
The role of CRP (which binds both to ChoP in the presence of calcium and to the first component of complement) in the antibody-independent complement activation
by the classical pathway was examined (22, 23). Removal
of CRP from the serum eliminated the difference in serum
sensitivity between the ChoP variants of strain H233 (Fig.
2 A). The role of CRP in the bactericidal activity of serum was confirmed by the addition of purified CRP to the
NHS depleted of CRP. The addition of CRP at a concentration of 5 µg/ml completely restored the increased serum
killing of the ChoP+ variant but had no effect on the
ChoP variant. CRP (5 µg/ml) added to bacteria in the
absence of serum or in serum pretreated at 56°C for 30 min
to remove complement activity had no effect on viability.
A dose response determination for the contribution of
CRP to serum bactericidal activity showed that the half-maximal effect on killing of the ChoP+ variant was at a
CRP concentration of 10 ng/ml (Fig. 3). The effect of
CRP was shown to require the presence of calcium. There was no increased killing of the ChoP+ variant when bacteria were pretreated with CRP (5 µg/ml) in 2 mM EDTA
in comparison to pretreatment in 10 mM Ca2+ (Fig. 2 B).
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Fig. 3.
The dose response to CRP in serum bactericidal assays. Survival of phase variants expressing (solid circles) or not expressing (open
squares) ChoP in 10% NHS depleted of CRP with purified human CRP
added at the concentration indicated was compared. The percentage of
survival is the number of CFUs remaining compared to controls in which
complement was inactivated.
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The direct interaction of purified CRP with strain H233
expressing ChoP and the requirement for calcium were
demonstrated by incubating both variants in CRP (5 µg/
ml) in the presence of Ca2+ (10 mM) or EDTA (2 mM)
(Fig. 4). After washing the cells to remove the unbound
CRP, the binding of CRP to whole bacteria was detected on Western blot analysis using an mAb to human CRP.
Binding of CRP required Ca2+ and only the ChoP-containing variant bound significant amounts of CRP. The
faint binding to the ChoP-variant in the presence of Ca2+
probably represents the <1% of ChoP+ revertants in this
population of organisms. The requirement of Ca2+ raised
the possibility that bactericidal assays in the presence of
0.05 M Mg2+ EGTA inhibited CRP binding rather than
the classical pathway of complement activation. However,
in bactericidal assays where bacteria were preincubated
with CRP in 10 mM Ca2+, chelation of divalent cations
did not remove bound CRP but eliminated killing by
complement. This confirmed that CRP-mediated killing requires complement activation by the classical pathway.
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Fig. 4.
The binding of human CRP to phase variants expressing ChoP in the presence of
calcium. Equivalent numbers of
phase variants with or without
ChoP were incubated in purified
CRP in the presence of Ca2+ or
EDTA. After removing the unbound CRP, the bound CRP
was detected on Western blot
analysis using an mAb that recognizes human CRP.
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The Contribution of ChoP in Nasopharyngeal Colonization of
Infant Rats.
The results of the bactericidal assays suggested
that the ChoP phenotype may be important in evading
humoral clearance mechanisms, but left unclear the contribution of the ChoP+ phenotype to the biology of H. influenzae. This question was addressed by comparing ChoP+
and ChoP variants in the infant rat model of nasopharyngeal colonization (Fig. 5). H. influenzae type b (strain
Eagan) was used in these experiments, since nontypable
isolates colonize poorly in animal models of carriage. After
an intranasal inoculum with 106 CFU of ChoP+ or variants of a population >97% the desired phenotype, the
number of organisms obtained from nasal washes remained
relatively constant for up to 16 d. Animals receiving the
ChoP+ variant remained colonized with organisms of this
phenotype (>99%) when assessed by colony immunoblotting. Those animals receiving the ChoP variant, in contrast, showed a gradual shift in the phenotype of organisms
cultured from the nasopharynx; at the final time point,
>73% of colonies had switched to the ChoP+ phenotype.
This finding suggests that decoration of the LPS with ChoP
was associated with an enhanced ability to persist within the nasopharynx. This result was confirmed by showing the
diminished ability of a lic1 mutant of strain Eagan with a
constitutively ChoP phenotype to persist in the infant rat
nasopharynx. By day 10 after the inoculation the lic1 mutant was present in significantly fewer numbers compared
to the ChoP+ variant. At day 16 after the inoculation, 8 out of 25 pups receiving the mutant had no detectable organisms in nasal washes versus 0 out of 22 receiving the
ChoP+ variant (P = 0.0034, Fisher's exact test).
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Fig. 5.
The contribution
of ChoP expression to H. influenzae carriage in an infant rat
model. ChoP variants of strain
Eagan and a lic1 mutant of this
strain were compared for their
ability to colonize the infant rat
nasopharynx. At each time point
the number and phenotype of
organism recovered in nasal
washes were determined. The
vertical axis represents the average number of organisms per animal in the inoculum or nasal
washes on the day after intranasal inoculation indicated (first bar at
each time point, ChoP+ inoculum; middle bar at each time
point, ChoP inoculum; last bar at each time point, lic1- mutant inoculum). Values are the geometric mean ± SD for at least 22 pups per variant or mutant in three separate experiments. The proportion of variants of the ChoP+ or ChoP phenotype in the inoculum or recovered in nasal washes at each time point were determined by colony immunoblotting and is indicated (ChoP+ solid portion; ChoP hatched portion). Colony immunoblotting was not
performed for the constitutive lic1 mutant (open bars).
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Factors Affecting the Expression of ChoP.
A number of
growth conditions were compared to determine whether
an environmental condition other than the availability of choline might account for the increase in the proportion of
organism of the ChoP+ phenotype in the nasopharynx.
The frequency of phase variation between phenotypes and
the incorporation of choline were compared after growth
to midlog phase in a chemically defined medium under various conditions including temperature (30-39°C), pH
(6.8-8.0), supplemental NaCl (0-1 M), Ca2+ (0.07-1.4
mM), Mg2+ (0.49-4.9 mM), or glucose (0.17-20 mM). Alternative sources of carbon (glycerol, pyruvate, lactate,
maltose, lactose, and galactose) at a concentration of 5 mM
in lieu of glucose were also examined. There was no significant difference for the ChoP+ variant in [3H]choline incorporation/milligrams of total cellular protein or in the
rate of reversion to the ChoP phenotype in colony immunoblots after growth under these conditions.
Chromosomal DNA extracted from organisms used to
inoculate rat pups was used in the PCR reaction to isolate
the 5-end of the lic1 locus to determine the number of repeats of 5-CAAT-3 within licA. As predicted the ChoP
variants had a number of repeats (n = 20) in which there
was no full-length translation product because no upstream
initiation codon was in frame with the licA gene (Fig. 6).
The ChoP+ variant, in contrast, had one fewer repeat (n = 19) which would place two potential initiation codons (labeled 
and 
) that are in the same reading frame, in the
correct orientation for translation of the full-length licA
gene product. Colonies obtained from nasal washes at day
10 after the inoculation were immunoblotted and used as a
source of DNA for amplification of this same region. In
each instance those colonies retaining the phenotype of the inoculum had the same number of repeats as those in the
inoculum. Organisms that switched from ChoP+ in the inoculum to ChoP at day 10 acquired an additional repeat
as predicted (n = 19 varied to n = 20). Organisms that
switched from ChoP in the inoculum to ChoP+ at day 10 either gained one (n = 21) or lost two repeats (n = 18).
Thus in four out of four colonies with organisms reverting to the ChoP+ phenotype, the number of repeats indicated
that licA was in frame with an initiation codon, although in
each case this was the 
and not the / initiation codons.
It was concluded that the only identifiable factor contributing to variation in expression of ChoP on the LPS was the
number of repeats of 5-CAAT-3 in licA.
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Fig. 6.
The correlation between ChoP
expression and the genotype of the lic1 locus. Shown above is the molecular mechanism controlling phase variation in expression of ChoP. The nucleotide sequence at
the 5 end of lic1 contains a variable number
(n) of tandem repeats of CAAT. Variation
in the number of repeats creates a translational switch with possible translation products in phase with the open reading frame
of licA indicated below the nucleotide sequence. Three potential initiation codons
(boxed) labeled , and are present in
only two of the three possible reading frames since and are positioned in the
same phase. (In some strains the codon is
GTG rather than ATG as shown.) Only
when an initiation codon is in frame with
the remainder of the open reading frame of
licA is there expression of ChoP. The table
on the bottom shows that ChoP expression
correlates with number of CAAT repeats
(n). Colonies obtained from nasal washes of
infant rats 10 d after the inoculation showed
a variant number of repeats only when the
phenotype in colony immunoblots was different from that of the corresponding inoculum.
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The Expression of ChoP in H. influenzae in the Human Respiratory Tract.
In the animal model there was an absolute
correlation between the phenotype of organisms with respect to ChoP and the number of repeats of CAAT in licA.
Based on this observation, the genotype of H. influenzae in
respiratory tract secretions from humans was determined in
order to deduce the in vivo phenotype (ChoP+ or ) of organisms within the natural host. Purulent or nonpurulent respiratory tract secretions were used as a source of DNA
for the PCR reaction to obtain the 5 end of lic1 for sequencing to determine the number of CAAT repeats. For
13 out of 14 specimens the number of repeats indicated
that the phenotype within the human respiratory tract was
ChoP+ (Table 2). As was demonstrated in the animal
model in instances of reversion to the ChoP+ phenotype,
there was a preference for a number of repeats that would
position the initiation codon in frame with licA (12 out of 14 for , 1 out of 14 for /). The only specimens in
which the number of repeats would not predict licA translation from the initiation codon were from purulent sputum that fulfilled the case definition of nontypable H. influenzae pneumonia (20).
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Table 2
Genotypic Analysis of the Number of CAAT Repeats (n)
in licA in Nontypable H. influenzae (NTHI) in Human Respiratory
Tract Secretions*
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Discussion |
We previously reported that one of the phase-variable
structures on the H. influenzae LPS included the ChoP
epitope. Structural analysis in this study confirms that variation in the display of this epitope corresponds to the presence or absence of the ChoP structure on the oligosaccharide of the LPS. Phase variants were then separated based
on the expression of ChoP as determined by reactivity with
mAbs recognizing this structure to address how ChoP contributes to the pathogenesis of infection caused by H. influenzae.
Since ChoP appears to be a common feature on the cell
surface of mucosal pathogens, particularly those residing in
the human respiratory tract, observations on H. influenzae
may be of relevance to the other species expressing the
ChoP structure/epitope (2, 12). This study was carried out
using H. influenzae because both the genetic and structural
basis of choline incorporation have now been defined in
this species (1, 5). We concentrated on nontypable isolates;
these organisms have been a predominant cause of disease
in adults for many years, and have become increasingly important in young children as H. influenzae type b disease has begun to disappear due to widespread vaccination. Phase
variation in ChoP expression made it possible to examine
bacteria with and without this characteristic in an identical
genetic background. An additional feature favoring the use
of H. influenzae in these experiments was that, unlike the
pneumococcus, H. influenzae does not appear to have multiple surface proteins anchored to ChoP (25). The absence
of such proteins in H. influenzae allowed us to examine the
direct contribution of ChoP to pathogenesis.
H. influenzae is found exclusively in the human nasopharynx, where it is able to persist for extended periods.
In this study, an animal model of H. influenzae carriage was
used to demonstrate a gradual selection for ChoP+ phase
variants during prolonged colonization of the nasopharynx. As predicted from this comparison of phase variants, a constitutively ChoP mutant was cleared from the infant rat
nasopharynx significantly more rapidly than from the wild-type controls. The capacity of ChoP+ variants to evade
clearance may help to explain why this structure may be
widely conserved among species that occupy a similar environment but are otherwise highly distinct. However, this
study does not address the specific mechanism by which
decoration of the LPS with ChoP allows H. influenzae to
persist in the nasopharynx. ChoP, which is also found on
eukaryotic membrane lipids, may provide a selective advantage on the mucosal surface through molecular mimicry
of the host. In tissue explants from the human nasopharynx, H. influenzae displays a tropism for the mucus layer
(26). Phospholipids related to phosphatidylcholine, which
encompass the ChoP structure, are present in mucus. No
contribution of the lic1 genes to the interaction of H. influenzae with human nasal turbinate tissue in culture was
demonstrated (26). However, these studies did not determine whether controls were phase on (ChoP+) or phase off
(ChoP). In addition, it has been suggested that ChoP
contributes to adherence of the pneumococcus to host cells
by binding to the receptor for platelet activating factor,
whose natural ligand also contains ChoP (27). If ChoP is
functioning as an adhesin in H. influenzae, we would predict that its contribution to colonization would be apparent
at an earlier stage after the intranasal challenge than was observed in the animal studies.
H. influenzae variants can be stratified into serum-resistant and serum-sensitive phenotypes based on the presence
of ChoP. The increased serum sensitivity of the ChoP+
variants has been previously shown to correlate with the
presence of anti-ChoP antibody in human serum (1). This
study demonstrates that the difference in serum sensitivity
is independent of antibody yet involves the classical pathway of complement activation. LPS structure is known to
affect the deposition of complement, but in previous reports this involved alterations of the O-antigen and was
mediated by the alternative pathway (28). The activation of
the classical pathway in the absence of antibody was shown to occur by the calcium dependent binding of CRP exclusively to the ChoP+ variant. CRP is capable of substituting
for attached antibody in activating the complement cascade
by the classical pathway through binding to C1q (29, 30).
Therefore, it appears that CRP, an acute phase reactant,
contributes to innate immunity to bacterial pathogens
other than the pneumococcus in which its role in host protection has long been postulated (31). In the case of the
pneumococcus, CRP is thought to promote clearance by
opsonization, whereas for H. influenzae CRP contributes to
killing through the bactericidal activity of complement.
The effect of CRP on serum bactericidal activity against H. influenzae strain H233 occurred at concentrations as low as
10 ng/ml. This is well below the level of CRP in normal,
unstimulated human serum (<200 ng/ml). A further implication of these results is that the innate immunity mediated by CRP may be particularly important in protection
against certain invasive mucosal pathogens, such as the
pneumococcus, meningococcus, and some H. influenzae
strains, in the time preceding emergence of antibody-mediated opsonizing and/or bactericidal activity.
We have noted that for H. influenzae, as well as for the
pathogenic Neisseria and the pneumococcus, there is variation between phenotypes with greater or lesser amounts of
the ChoP structure/epitope (references 1 and 14 and Weiser,
J.N., J. Goldberg, N. Pan, and M. Virgi, manuscript submitted for publication). This suggests that there may be a
role for both these phenotypes in the interaction of ChoP-expressing pathogens and their host. H. influenzae has the
ability to switch off expression of ChoP, which eliminates binding of CRP and the subsequent activation of complement through the classical pathway. This suggests that the
biological role of the ChoP phenotype might be evasion
of CRP-mediated clearance. It is predicted that there
would be a selection for the ChoP variants from among
the predominantly ChoP+ population in the commensal
state whenever the concentration of CRP and complement
is sufficient. This may occur during invasive infection, localized inflammation, or systemic inflammatory states when
serum levels of CRP rise precipitously. It was not possible to demonstrate a selection for ChoP variants among the
infant rats developing bacteremia after the intranasal inoculation. Unlike humans, CRP expression in rats is at constitutively low levels for the first 15 d after gestation (32). The
30-fold increase in CRP levels in normal adult rats may
also account for the age-related sensitivity of this model host to invasive infection by H. influenzae.
The mechanism-controlling phase variation in ChoP expression provided a means of determining the phenotype of
organisms without the need for in vitro culture (11).
Avoiding in vitro growth was desirable because of the high
rate of phenotypic switching. In preliminary experiments,
we demonstrated that the only factor which affected phase
variation was the number of repeats of CAAT within the
lic1. In each of eight phenotypic revertants obtained from
nasal washes of infant rats, there was a corresponding shift in the number of repeat units. The phenotype of organisms
in the respiratory tract of the natural host was then deduced
by determining the genotype with respect to lic1. If ChoP
contributes to persistence in the nasopharynx, organisms in
respiratory tract secretions at any point in time would tend
to have ChoP that contains LPS. As expected, the overwhelming majority (92.9%) of patients with H. influenzae
identified in their respiratory secretions had a number of
repeats which would predict translation of the full length
licA gene and expression of the ChoP+ phenotype. This
finding supports the hypothesis that the ChoP decoration
of the LPS contributes to the ability of H. influenzae to colonize and persist within the human respiratory tract, the initial step in the pathogenesis of infection.
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