Molecular basis of increased serum resistance among pulmonary isolates of non-typeable Haemophilus influenzae

Abstract

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies.

Figure 1. Characterization of clinical isolates.

(A) Comparison of serum sensitivity between lower and upper respiratory tract isolates. Survival was determined over 60 min in 5% normal human serum and expressed relative to controls in which complement was inactivated. Groups included lower respiratory tract isolates from patients with chronic obstructive pulmonary disease (COPD) at the time of clinical exacerbation (black bars; n = 11); isolates from patients with COPD during clinically stable periods (grey bars; n = 11); and upper respiratory tract colonizing strains (white bars; n = 25). Values are the mean of three determinations in triplicate ± SEM. (B) Comparison of antibody binding in serum resistant (>50% survival in pooled NHS, black bars; n = 10) and serum sensitive (<50% survival in pooled NHS, white bars; n = 10) isolates. B1 and B2 show percent of IgG and IgM bound following incubation in 5% heat-inactivated NHS as determined by flow cytometry, respectively. (C) Serum IgM contributes to bactericidal killing of clinical isolates of NTHi. Strains were incubated with or without IgG (C1, 0.25 µg/ml) or IgM (C2, 0.07μg/ml) purified from NHS for 60 min in 2.5% baby rabbit serum as a complement source. Percent survival was calculated by viable counts with and without antibody. The mean values of two independent experiments in triplicate are shown ± SD, ^* P<0.05, ^** P<0.01, ^*** P<0.001. Serum sensitive (SS), Serum resistant (SR).

Figure 2. Characterization of vacJ and yrb mutants.

(A) Effect of mutations in vacJ and genes of the yrb ABC transporter on serum resistance of strain R2866. Survival was determined over 60 min in 5% normal human serum and expressed relative to controls in which complement was inactivated. (B) Representative histogram comparing the binding, as measured by fluorescence intensity (x-axis), of total IgM purified from normal human serum to parent strain (WT) or vacJ by flow cytometry. Control performed without IgM (C) Percent IgM binding for each mutant was determined by calculating the percentage of 50,000 events with an increase in mean fluorescence intensity following incubation in 5% heat-inactivated normal human serum compared to no serum controls. (D) Survival of mutants in 10% normal human serum in the presence (black bars) or absence (white bars) of Mg-EGTA to inhibit the classical pathway of complement activation. Values represent two independent experiments in triplicate ± SD. ^* P<0.05, ^** P<0.01, ^*** P<0.001.

Figure 3. Effect of vacJ and yrb mutants on antibody binding and bactericidal activity.

Binding of (A) mAb 4C4 to the LOS structure Galα1-4Gal or (B) mAb TEPC-15 to the LOS structure phosphorylcholine was compared by flow cytometry for the mutants indicated. Percent mAb binding for each mutant was determined by calculating the percentage of 50,000 events with an increase in mean fluorescence intensity compared to no primary antibody controls. (C) Effect of mutations in vacJ and genes of the yrb ABC transporter on the bactericidal effect of mAb 4C4. Bactericidal assays were performed with (white bars) or without (black bars) mAb with 5.0% normal mouse serum as a complement source. Values represent two independent experiments in triplicate ± SD. ^* P<0.05, ^*** P<0.001.

Figure 4. Effect of mutations in vacJ and genes of the yrb ABC transporter on outer membrane characteristics.

(A) To compare outer membrane stability, following overnight culture of the strain indicated, viable counts were obtained after incubation at 37°C for 4 h in the presence (white bars) or absence (black bars) of 25 mM EDTA. Values represent two independent experiments in triplicate ± SD. ^* P<0.05. (B) To compare surface hydrophobicity, the rate of uptake of membrane permeant 1-N-phenylnaphthylamine (NPN) was monitored by fluorescence. NPN was added at the time indicated and a representative experiment shown. (C) To compare amounts of surface phospholipids, diacylglycerol (boxed area) released by phospholipase C treatment of whole bacteria was detected by thin layer chromatography. Lane 1; diacylglycerol (standard), Lane 2 and 3; R2866 (wild type), Lane 4 and 5; 32F2 (vacJ mutant), Lane 6 and 7; 69G3 (yrbE mutant).

Figure 5. Membrane stability and vacJ and yrb expression among clinical isolates.

(A) Outer membrane stability of serum sensitive (n = 31) compared to serum resistant (n = 16) clinical isolates. Following overnight culture, viable counts were obtained after incubation at 37°C for 4 h with or without 25 mM EDTA. Values represent two independent experiments in triplicate ± SD. ^* P<0.05, Serum sensitive (SS), Serum resistant (SR). (B) Relative expression of vacJ mRNA, (C) yrbD and (D) yrbE by qRT-PCR in serum sensitive (white bar, n = 7) and serum resistant isolates (black bar, n = 7). Error bars indicate SD, ^** P<0.01.

Figure 6. Repeated serum treatment selects for serum resistance, increased vacJ expression, and increased outer membrane stability.

(A) Serum sensitive clinical isolate H725 was treated three times in 2.5% NHS and survival quantified after the passage indicated. (B) Following each passage in the bactericidal assay survivors were tested for relative expression of vacJ mRNA by qRT-PCR and (C) survival in the presence of 25 mM EDTA. Values represent two independent experiments in triplicate ± SD.^* P<0.05, ^*** P<0.001.