RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signalling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2.

Figure 1. AI-2 accumulates in late-exponential phase cultures of NTHI 86-028NP rbsB::Cm

NTHI 86-028NP, NTHI 86-028NP rbsB::Cm, and the complemented strain NTHI 86-028NP rbsB::Cm IRB::Sp were cultured in sBHI to stationary phase and supernatant samples were removed and stored at −20°C. Determination of AI-2 production in thawed supernatants was performed using the Vibrio harveyi bioluminescence assay. Significance was determined by a two-way ANOVA with a post-hoc test. *P<0.05. Error bars indicate mean and standard deviation for three independent experiments and duplicate samples for bioluminescence.

Figure 2. Mutation of rbsB limits AI-2 depletion

NTHI 86-028NP luxS::Kn and NTHI 86-028NP rbsB::Cm luxS::Kn were cultured in sBHI media alone or sBHI supplemented with 0.2 μM of the chemically synthesized AI-2 precursor (S)-4,5-Dihydroxy-2,3-pentanedione (DPD) and samples were taken to assess depletion of DPD by V. harveyi bioluminescence. An uninoculated control sample of sBHI supplemented with DPD was included as a negative control for depletion. (A) A representative graph showing kinetics of AI-2 depletion by NTHI 86-028NP luxS::Kn and NTHI 86-028NP rbsB::Cm luxS::Kn. Error bars indicate mean and standard deviation for duplicate samples. (B) Combined results of five independent experiments with duplicate samples showing the percent of AI-2 depleted by NTHI 86-028NP luxS::Kn versus NTHI 86-028NP rbsB::Cm luxS::Kn for culture supernatants taken at an OD₆₀₀ of ~0.85, normalized to uninoculated control samples. Error bars indicate mean and standard deviation.

Figure 3. AI-2 increases transcription of luxS and rbsB

NTHI 86-028NP and NTHI 86-028NP luxS::Kn were cultured in sBHI media to stationary phase. Samples were taken during lag phase (OD₆₀₀ 0.200), exponential phase (OD₆₀₀ 0.500), late-exponential phase (OD₆₀₀ 0.750), and early stationary phase (OD₆₀₀ 0.950) for isolation of RNA and real time RT-PCR analysis of luxS (A) and rbsB (B) transcript levels. Values represent the ratio of luxS or rbsB to gyrA transcript. Error bars indicate mean and standard deviation for duplicate samples. (C) NTHI 86-028NP luxS::Kn was cultured in sBHI to an OD₆₀₀ of ~0.650 and supplemented with either 0.2 μM DPD or sterile water. Samples were taken prior to the addition of DPD or water and at 10, 30, 40, 50, and 90 minutes afterwards for RNA isolation and real time RT-PCR analysis of rbsB transcript levels. Values represent the fold increase in rbsB transcript (normalized to gyrA) for the DPD treated samples compared to untreated samples. Error bars indicate mean and standard deviation for duplicate sampling from two independent experiments.

Figure 4. Mutation of rbsB results in a similar biofilm defect as mutation of luxS

Biofilms formed by NTHI 86-028NP, NTHI 86-028NP luxS::Kn, and NTHI 86-028NP rbsB::Cm under continuous flow conditions were visualized with a live/dead stain by CLSM. Z-series images were used to create representative volume views of biofilms formed by NTHI 86-028NP (A), NTHI 86-028NP luxS::Kn (B), and NTHI 86-028NP rbsB::Cm (C). Z-series images were also exported to COMSTAT to obtain biofilm measurements, including total biomass (D), surface to biovolume ratio (E), average biofilm thickness (F), and maximum biofilm thickness (G). Statistical significance determined by unpaired t test, error bars indicate standard error of the mean (n=8).

Figure 5. Genetic complementation fully restores biofilm formation by NTHI 86-028NP rbsB::Cm

NTHI 86-028NP, NTHI 86-028NP luxS::Kn, NTHI 86-028NP rbsB::Cm, and the complemented strain NTHI 86-028NP rbsB::Cm IRB::rbsB were cultured in sBHI media and allowed to establish stationary biofilms for 12 h. Biofilms were visualized by live/dead staining and CLSM. Vertical z-series images were used to create representative volume views of biofilms formed by NTHI 86-028NP (A), NTHI 86-028NP rbsB::Cm IRB::rbsB (B), NTHI 86-028NP rbsB::Cm (C), and NTHI 86-028NP luxS::Kn (D). Z-series images were also exported to COMSTAT to obtain measurements of total biofilm biomass (E) and average thickness (F). Statistical significance determined by unpaired t test. Error bars indicate mean and standard deviation (n=6).

Figure 6. NTHI 86-028NP requires rbsB to respond to exogenous AI-2

NTHI 86-028NP, NTHI 86-028NP luxS::Kn, and NTHI 86-028NP rbsB::Cm were cultured in sBHI media alone (white bars) or sBHI supplemented with 0.2 μM DPD (gray bars) and allowed to establish stationary biofilms for 12 h. Biofilms were visualized by live/dead staining and CLSM. Vertical z-series images were exported to COMSTAT for analysis of total biofilm biomass (A) and average thickness (B). Statistical significance determined by unpaired t test. **P<0.01 compared to NTHI 86-028NP. ^*P<0.01 compared to NTHI 86-028NP luxS::Kn. Error bars indicate mean and standard deviation (n=6).

Figure 7. Mutation of rbsB reduces surface-accessible PCho

A modified whole-bacterium ELISA was used to detect surface phosphorylcholine using an anti-PCho monoclonal antibody. Significance determined by unpaired t test, error bars indicate standard error of the mean (n=12).

Figure 8. Mutation of rbsB limits bacterial persistence in vivo

Chinchillas were infected via transbullar inoculation with ~10³ cfu of NTHI 86-028NP (filled circles), NTHI 86-028NP luxS::Kn (open triangles), or NTHI 86-028NP rbsB::Cm (open diamonds). Total bacterial load per ear was determined by serial dilution and plating of effusion fluid, middle ear lavage, and bullar homogenate at 7, 14, 21, and 28 days post-infection. Data points represent total bacterial load for individual ears, and data shown are the combined results for three independent studies (two 21-day studies and one 28-day study). Day 7: NTHI 86-028NP n=15 ears, NTHI 86-028NP luxS::Kn n=17 ears, NTHI 86-028NP rbsB::Cm n=15 ears. Day 14: NTHI 86-028NP n=15 ears, NTHI 86-028NP luxS::Kn n=15 ears, NTHI 86-028NP rbsB::Cm n=15 ears. Day 21: NTHI 86-028NP n=14 ears, NTHI 86-028NP luxS::Kn n=16 ears, NTHI 86-028NP rbsB::Cm n=16 ears. Day 28: NTHI 86-028NP n=8 ears, NTHI 86-028NP luxS::Kn n=6 ears, NTHI 86-028NP rbsB::Cm n=8 ears. The dashed line indicates limit of detection. Error bars represent the geometric mean and 95% confidence intervals. Significance determined by log transformation of the data and unpaired t test.