Exploiting the struggle for haem: a novel therapeutic approach against Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a common coloniser of the upper respiratory tract in healthy children (20–80%) and adults (20–30%), the prevalence of which varies considerably across geographical regions^1–4. However, in susceptible individuals, NTHi represents a major cause of opportunistic infections in the respiratory tract, namely acute otitis media and sinusitis in children, and lower respiratory tract infections in elderly individuals or those with chronic obstructive pulmonary disease⁵. Collectively, these infections and subsequent long-term health complications, such as hearing loss or decline in lung function, impart a significant global disease burden^5,6. Further amplifying the global impact of NTHi infections is the rapidly expanding spectrum and prevalence of antibiotic resistance, leading to treatment failure with first- and second-line antibiotics^5,7. The high morbidity and long-term antibiotic prescription associated with NTHi infections, collectively expose a substantial proportion of the population to antimicrobial agents, driving resistance to a broad-spectrum of antibiotics in the community^8,9. The threat of antibiotic resistance was recognised by the World Health Organization in 2017, listing NTHi as a priority pathogen for which new therapies are urgently needed¹⁰. Owing to the high genetic heterogeneity and phase-variable expression of conserved antigen targets, there are currently no effective vaccine strategies available that can slow the growing burden of NTHi disease¹¹. Consequently, novel preventative or therapeutic approaches that do not rely on antibiotic susceptibility or stable vaccine targets are becoming more attractive.

The pathogenesis of NTHi is largely dictated by interactions with host airway epithelia. Although the exact mechanisms are poorly understood, NTHi adhesion and colonisation of the host pharyngeal epithelium, followed by migration to privileged anatomical sites, is required to elicit an infection¹². Survival and persistence at the site of infection is mediated by host-cell internalisation, formation of biofilms, or modulation of the immune response that protects bacterial populations from immune or antibiotic clearance^13–15. In addition to being an essential growth requirement, access to iron-containing haem plays an important role in the ability of NTHi to perform these interactions and as such, the ability to sequester host-derived sources of haem is a key determinant of pathogenesis^16,17. The consequence of NTHi haem starvation, either by disruption of acquisition mechanisms or by environmental restriction, has been demonstrated to attenuate virulence in animal models of invasive disease and otitis media^18–21. Strategies that interrupt NTHi acquisition or utilisation of host-derived sources of haem may therefore have a significant impact on the ability of NTHi to cause disease.

Recently, we discovered strains of the closely related commensal Haemophilus haemolyticus (Hh) that also inhabit the pharyngeal niche and secrete a novel haemophore (since named haemophilin; Hpl) that elicits potent inhibitory activity against NTHi^22,23. Functional and proteomic investigation demonstrated that Hpl is a previously unrecognised haem uptake mechanism of Hh, which inhibits NTHi growth through exploitative competition for haem. We have since conducted several investigations in vitro and in vivo to test the NTHi-inhibitory capacity of Hpl-producing strains of Hh (Hh-Hpl⁺) and propose their therapeutic utility as a respiratory probiotic.

In a broth co-culture system, NTHi strains were outcompeted by Hh-Hpl⁺ and suffered a complete loss of fitness over subsequent generations²⁴. Similarly, in tissue culture models of nasopharyngeal (D562) and lung epithelia (A549), Hh strains with high levels of hpl expression protected cell monolayers against adhesion and invasion by NTHi²⁵ (Figure 1). Significant inhibition of NTHi adherence and invasion was maintained when Hh-Hpl⁺ treatment doses were 10–100-fold lower than the NTHi challenge. In both in vitro models, NTHi-inhibitory activity correlated with levels of hpl expression and Hpl protein quantified from competition media. The absence of NTHi-inhibitory activity in a hpl knockout or native non-producing strains confirmed that the inhibitory phenotype was mediated by the ability to produce Hpl.

Figure 1.  NTHi attachment and invasion of A549 and D652 cells post treatment with Haemophilus haemolyticus (Hh) strains (BW1, RHH122, NF5, NF5, NF1) or the hpl knockout (BW1^hpl-KO). The percent attachment of NTHi (compared to media control) to A549 (a) and D562 (b) cell monolayers post 4-h pre-treatment with Hpl-producing Hh (Hh-Hpl⁺) or Hh strains that do not produce Hpl (Hh-Hpl^–). Percent of internalised NTHi (compared to media control) after exposure to A549 (c) and D562 (d) cell monolayers post 4-h pre-treatment with Hh-Hpl⁺ or Hh-Hpl^–. Error bars represent the ±SEM (standard error of the mean) of three biological replicates, measured triplicate: *P < 0.05, ****P < 0.0001.

Considering the NTHi-inhibitory activity in vitro we hypothesised that natural pharyngeal carriage of Hh strains with the hpl open reading frame would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl⁺ or Hh-hpl^– genotype from 257 healthy adults in Australia. Compared to carriage of Hh-hpl^– strains, adult (18–65 years) and elderly (>65 years) participants that were colonised with Hh-hpl⁺ were 2.43 (95% CI, 1.95–2.61; P < 0.0001), or 2.67 times (95% CI, 2.63–2.70; P = 0.0036) less likely to carry NTHi, respectively. Colonisation with high densities of Hh-hpl⁺ correlated with low NTHi carriage load and a 2.63-times (95% CI, 2.56–2.70, P = 0.0112) lower likelihood of acquiring/maintaining NTHi colonisation status between visits²⁶ (Figure 2).

Figure 2.  NTHi dominance in oropharyngeal swabs of healthy adult (18–65 years) or elderly (>65 years) participants co-colonised with Hh. NTHi oropharyngeal carriage prevalence (a) or proportion of NTHi (as a function of total Hh) (b) among participants concurrently carrying Hh strains that possess the hpl ORF (Hh-hpl⁺) or do not possess the hpl ORF (Hh-hpl^–). Hh-hpl⁺ (predominant) denotes instances where hpl⁺ is the predominant Hh genotype (>0.5 of total Hh). NTHi colonisation status in participants carrying hpl⁺ (n = 25) or hpl^– (n = 25) strains of Hh on follow-up testing (visit 2) 2–6 months after their initial visit (visit 1). Error bars represent ±SEM (standard error of the mean); statistical significance was determined by simple logistic regression (a) or nonparametric Spearman correlation (b); **P < 0.005, ***P < 0.001, ****P < 0.0001.

The presence of healthy carriers of NTHi indicates that a complete eradication of NTHi is not necessary to prevent infection. Furthermore, higher NTHi pharyngeal carriage loads are correlated with an increased susceptibility to otitis media in vivo^27–30 and an increased severity of airway inflammation, exacerbations, and daily symptoms in chronic obstructive pulmonary disease^31,32. Thus, even small reductions in NTHi carriage might have beneficial clinical outcomes. Using a model designed to predict the risk of otitis media in children based on NTHi pharyngeal carriage load³⁰, we could predict a ≈40% decrease in the risk of infection, provided the level of protection conferred by Hpl-producing Hh to model cell lines was preserved in the context of the respiratory tract. Hh also possesses favourable characteristics suited to probiotic applications; it has not been implicated as a causative agent of respiratory tract infection^33,34 and as a normal pharyngeal inhabitant, is able to thrive in the niche amongst other microbial inhabitants³⁵. Additionally, probiotic-based therapies have a narrow spectrum of activity that do not damage host tissue, provoke collateral damage to the healthy microbiome or promote enrichment of resistant clones³⁶; properties which make them an asset against the emergence of antibiotic resistance.

In conclusion, Hpl-producing Hh may be a promising respiratory probiotic candidate for the prevention of NTHi infections by inhibiting requisite pharyngeal colonisation.

The authors declare no conflicts of interest.

The studies informing this work were funded by the Clifford Craig Medical Research Trust (CCF170, CCF192).