Phosphorylcholine on the Lipopolysaccharide of Haemophilus influenzae Contributes to Persistence in the Respiratory Tract and Sensitivity to Serum Killing Mediated by C-reactive Protein

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: δ 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.

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. ​(Fig.22 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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Figure 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.

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. ​(Fig.22 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. ​(Fig.3).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. ​(Fig.22 B).

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Figure 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.

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. ​(Fig.4).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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Figure 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.

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. ​(Fig.5).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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Figure 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).

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. ​(Fig.6).6). The ChoP⁺ variant, in contrast, had one fewer repeat (n = 19) which would place two potential initiation codons (labeled α and β) 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 γ and not the α/β 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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Figure 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 α, β and γ are present in only two of the three possible reading frames since α and β are positioned in the same phase. (In some strains the β 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.