Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein - PubMed (original) (raw)

Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein

J N Weiser et al. J Exp Med. 1998.

Abstract

Haemophilus influenzae undergoes phase variation in expression of the phosphorylcholine (ChoP) epitope, a structure present on several invasive pathogens residing in the human respiratory tract. In this study, structural analysis comparing organisms with and without this epitope confirmed that variants differ in the presence of ChoP on the cell surface-exposed outer core of the lipopolysaccharide. During nasopharyngeal carriage in infant rats, there was a gradual selection for H. influenzae variants that express ChoP. In addition, genotypic analysis of the molecular switch that controls phase variation predicted that the ChoP+ phenotype was predominant in H. influenzae in human respiratory tract secretions. However, ChoP+ variants of nontypable H. influenzae were more sensitive to the bactericidal activity of human serum unrelated to the presence of naturally acquired antibody to ChoP. Serum bactericidal activity required the binding of C-reactive protein (CRP) with subsequent activation of complement through the classical pathway. Results of this study suggested that the ability of H. influenzae to vary expression of this unusual bacterial structure may correlate with its ability both to persist on the mucosal surface (ChoP+ phenotype) and to cause invasive infection by evading innate immunity mediated by CRP (ChoP- phenotype).

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Figures

Figure 1

Figure 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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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).

Figure 6

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.

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