Nontypeable Haemophilus influenzae newly released (NRel) from biofilms by antibody-mediated dispersal versus antibody-mediated disruption are phenotypically distinct

Abstract

Biofilms contribute significantly to the chronicity and recurrence of bacterial diseases due to the fact that biofilm-resident bacteria are highly recalcitrant to killing by host immune effectors and antibiotics. Thus, antibody-mediated release of bacteria from biofilm residence into the surrounding milieu supports a powerful strategy to resolve otherwise difficult-to-treat biofilm-associated diseases. In our prior work, we revealed that antibodies directed against two unique determinants of nontypeable Haemophilus influenzae (NTHI) [e.g. the Type IV pilus (T4P) or a bacterial DNABII DNA-binding protein, a species-independent target that provides structural integrity to bacterial biofilms] release biofilm-resident bacteria via discrete mechanisms. Herein, we now show that the phenotype of the resultant newly released (or NRel) NTHI is dependent upon the specific mechanism of release. We used flow cytometry, proteomic profiles, and targeted transcriptomics to demonstrate that the two NRel populations were significantly different not only from planktonically grown NTHI, but importantly, from each other despite genetic identity. Moreover, each NRel population had a distinct, significantly increased susceptibility to killing by either a sulfonamide or β-lactam antibiotic compared to planktonic NTHI, an observation consistent with their individual proteomes and further supported by relative differences in targeted gene expression. The distinct phenotypes of NTHI released from biofilms by antibodies directed against specific epitopes of T4P or DNABII binding proteins provide new opportunities to develop targeted therapeutic strategies for biofilm eradication and disease resolution.

Keywords: Antibiotic; Chronic infection; Dispersal; Disruption; Proteomics; Quorum sensing.

Fig. 1

Quantitation of NTHI released from biofilm-residence by either anti-rsPilA or anti-IHF. NTHI biofilms established for 16 ​h were incubated for an additional (a) 6 ​h with rabbit anti-rsPilA IgG or (b) 15 ​min with rabbit anti-IHF IgG or with each of three negative controls (sBHI, IgG isolated from naive serum or IgG isolated from anti-OMP P5 serum) followed by quantitation of NTHI recovered from supernatants above the biofilms. Anti-rsPilA and anti-IHF induced significant release of NTHI from biofilm residence into the NRel state. Individual data points are shown, bars represent mean ​± ​SEM.∗∗∗∗, P ​< ​0.0001, One-way analysis of variance with the Holm-Sidak correction.

Fig. 2

Release of NTHI from a biofilm by incubation with anti-rsPilA or anti-IHF antibodies generated NRel populations with distinct proteomic expression profiles compared to planktonically grown NTHI and, importantly, to each other. (a) Principal component analysis (PCA) plot generated from the normalized spectral counts of each protein in anti-rsPilA NRel (purple dots), anti-IHF NRel (orange dots) and planktonically grown NTHI (black dots). Triplicate samples of each population are encircled by 95% confidence ellipses. The proteomic expression profiles of anti-rsPilA and anti-IHF NRel were distinct from both planktonically grown NTHI, and from each other. (b) Venn diagram of the number of proteins with a significant (P ​< ​0.05) 1.5-fold increase (above the dashed line) or decrease (below the dashed line) specific to anti-rsPilA (purple), anti-IHF (orange), or shared (gray), compared to planktonic NTHI. (c) The anti-rsPilA (purple bars) and anti-IHF (orange bars) NRel demonstrated distinct protein expression patterns with a significant (P ​< ​0.05) 1.5-fold increase or decrease represented by different COG categories when compared to planktonically grown NTHI. (d) Direct comparison of differences in protein expression profiles of anti-IHF and anti-rsPilA NRel populations with a significant (P ​< ​0.05) 1.5-fold increase or decrease compared to each other, as shown by a volcano plot of anti-IHF NRel versus anti-rsPilA NRel. Negative significant fold decreases represent proteins with greater abundance in the anti-rsPilA NRel (purple dots), while positive fold increases represent greater protein abundance in the anti-IHF NRel (orange dots) compared to each other. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

NRel NTHI populations were more sensitive to killing than their planktonic counterparts, and this sensitivity was distinct from each other. (a) Diagram of the four populations of NTHI tested herein. NRel were generated by incubation of NTHI biofilms with rabbit polyclonal IgG isolated from anti-rsPilA serum (6 ​h, purple) or from anti-IHF serum (15 ​min, orange). (b&c) Anti-rsPilA NRel were significantly more sensitive to killing by trimethoprim/sulfamethoxazole than planktonic NTHI (TMP-SMX at 0.94 ​μg and 4.7 ​μg per ml respectively, panel b), but only equally as sensitive to killing by amoxicillin/clavulanate (AMC at 2.5 ​μg and 1.25 ​μg per ml, respectively panel c). Biofilm-resident NTHI displayed minimal sensitivity to either TMP-SMX or AMC as expected. (d&e) In contrast, anti-IHF NRel were only equally as sensitive to killing by TMP-SMX as planktonic NTHI (0.09 and 0.45 ​μg/ml respectively, panel d), but significantly more sensitive to killing by AMC (0.30 and 0.15 ​μg/ml respectively, panel e). The uniquely heightened sensitivity of NTHI NRel to killing by either TMP-SMX or AMC was dependent upon the mechanism by which they were released from biofilm residence. Individual data points are shown, bars represent mean ​± ​SEM. ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001, one-way analysis of variance with the Holm-Sidak correction. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Enhanced sensitivity of anti-rsPilA or anti-IHF NRel NTHI to TMP-SMX or AMC, respectively, was independent of the timing of NTHI release from biofilm residence. We exposed biofilms to rabbit polyclonal IgG isolated from either anti-rsPilA or anti-IHF serum for 2 ​h, then collected NRel NTHI and assayed for relative antibiotic sensitivity. (a&b) Anti-rsPilA NRel were significantly more sensitive to killing by TMP/SMX than planktonic NTHI (TMP-SMX at 0.09 ​μg and 0.45 ​μg per ml respectively, panel a), but only equally as sensitive to killing by AMC (AMC at 0.3 ​μg and 0.15 ​μg per ml, respectively panel b). (d&e) In contrast, anti-IHF NRel were only equally as sensitive to killing by TMP-SMX as planktonic NTHI (0.94 and 4.7 ​μg/ml respectively, panel c), but significantly more sensitive to killing by AMC (2.5 and 1.25 ​μg/ml respectively, panel d). These data showed that time-matched anti-rsPilA or anti-IHF NRel maintained the same distinct antibiotic sensitivity phenotype as shown when 15 ​min anti-IHF NRel were compared to 6 ​h anti-rsPilA NRel (see Fig. 3). Individual data points are shown, bars represent mean ​± ​SEM. ∗∗∗∗P ​< ​0.0001, one-way analysis of variance with the Holm-Sidak correction.

figs1

Anti-rsPilA NRel were released from a biofilm as individual cells whereas anti-IHF NRel were aggregated. (a) Contour plots depicted side scatter and forward scatter profiles for two control samples, NTHI briefly sonicated to produce an individual cell suspension or NTHI colonies collected from an agar plate to represent bacterial aggregates, assessed by flow cytometry. Representative contour plots of (b) anti-rsPilA NRel and (c) anti-IHF NRel demonstrated unique scatter profiles between the two NRel populations. The distribution of anti-IHF NRel (orange histogram) versus anti-rsPilA NRel (purple histogram) by (d) forward scatter and (e) side scatter was also distinct. The percent increase in cumulative distribution of anti-IHF NRel compared to anti-rsPilA NRel shown in (d) and (e) was determined by Kolmogorov-Smirnov test (99% CI). These data represented an additional discriminative characteristic of NRel populations generated via exposure to either anti-rsPilA or anti-DNABII antibody-mediated release of NTHI from biofilm residence.

figs2

Differences in relative gene expression support the observed distinct anti-IHF and anti-rsPilA NRel phenotypes, including antibiotic sensitivities. Results of qRT-PCR assay to examine the expression of genes by NRel relative to planktonic NTHI. (a) Relative expression of deaD, artM, and fis, genes associated with lag phase of growth, was significantly greater in anti-IHF vs. anti-rsPilA NRel. (b) The enzymes targeted by TMP and SMX are encoded by folA and folP respectively, and thereby increased expression confers resistance. Relative expression of folA and folP by anti-rsPilA NRel was significantly less than by anti-IHF NRel. (c) Relative expression of emrA and emrB, which encode subunits of an efflux pump that transports TMP-SMX, was significantly less by anti-rsPilA vs. anti-IHF NRel. Expression of acrR, which represses the efflux pump that transports AMC, was significantly elevated in anti-IHF vs. anti-rsPilA NRel. These patterns of relative gene expression support the enhanced sensitivities of anti-rsPilA or anti-IHF NRel to TMP-SMX or AMC, respectively. ∗P<0.05, ∗∗∗P<0.001, ∗∗∗∗P<0.0001, Student’s t-test