Potential therapeutic value of normal throat bacteria that produce a novel inhibitory substance for the prevention of respiratory infections

Nontypeable Haemophilus influenzae (NTHi) is a major opportunistic pathogen that causes a variety of infections in the respiratory tract, including community-acquired pneumonia, acute exacerbations of chronic obstructive pulmonary disease and otitis media. Collectively, these infections and subse-quent complications impose a significant global burden of disease. The impact of NTHi-associated disease is further amplified by the rapidly expanding spectrum and prevalence of antibiotic resistance, and the lack of an effective vaccination strategy. Consequently, novel preventative or therapeutic approaches that do not rely on antibiotic susceptibility or stable vaccine targets are becoming more attractive. One such approach may involve exploiting the bacterium‚ÄövÑv¥s nutritional dependency for host-derived iron-containing haem, the acquisition of which is a major determinant of NTHi survival and pathogenesis within the respiratory tract. Strategies that interfere with the acquisition of this essential nutrient may therefore have a significant impact on the ability of NTHi to cause disease. The limited availability and nutritional demand for haem-iron promotes a highly competitive environ-ment between NTHi and other upper respiratory tract inhabitants. We therefore hypothesised that an upper respiratory commensal capable of outcompeting NTHi for haem-iron may have utility as a probiotic therapy by generating an environment inhospitable for NTHi growth. Recently, nasopharyn-geal isolates of the closely related commensal Haemophilus haemolyticus (Hh), were discovered with the capacity to inhibit NTHi growth by secretion of a novel bacteriocin-like substance (herein referred to as haemophilin; Hpl). Hpl was found to possess structural characteristics consistent with that of a haem-binding protein and thus, we proposed a model by which Hpl inhibits NTHi growth by restricting access to haem. In Chapter 3, this hypothesis was tested by generating and comparing the NTHi-in-hibitory capacity of an hpl knockout to the wild-type strain under varying concentrations of haem or recombinant Hpl. These experiments indicated that Hpl was only inhibitory under haem-limited con-ditions. The loss of NTHi-inhibitory activity in media recovered from the knockout strain, and the di-rect correlation between NTHi-inhibitory activity and hpl expression across different Hh strains, con-firmed that Hpl was the primary mediator of NTHi growth inhibition by Hh. In addition to the loss of NTHi-inhibitory capacity, the knockout also displayed poor growth on haem supplemented media compared to the wild-type strain, indicating a defect in haem utilisation and a role for Hpl in Hh haem acquisition/utilisation. Growth of NTHi and Hh in media supplemented with either Hpl or the equiva-lent concentration of free haem demonstrated that Hpl-bound haem is available to Hh as a nutritional source of haem, but not to NTHi. To further characterise the biological role and genetic determinants of Hpl production, whole genome sequences of Hh isolates containing the hpl open reading frame (n=24) were generated. Among all isolates and publicly available genomes, the hpl gene occurred in a conserved gene cassette containing genes for a putative secretion protein and a putative Hpl/haem receptor. Together with supporting proteomic investigations, these findings provided evidence that Hpl has a role in haem acquisition by Hh, and that NTHi-inhibitory activity occurs through haem star-vation. We therefore proposed the possibility that competition from Hpl-producing Hh (Hh-Hpl\\(^+\\)) could antagonise NTHi colonisation in the respiratory tract. The feasibility of this approach was tested in chapter 4 by direct in vitro competition assays between NTHi and Hh strains with varying capacities to produce Hpl. Subsequent changes in NTHi growth rate and fitness, in conjunction with hpl expression analysis, were employed to assess the NTHi-inhibitory capacity of Hh strains. The growth rate of NTHi was significantly impaired during co-culture with Hh strains containing hpl (Hh-hpl\\(^+\\)), but not with strains lacking the hpl open reading frame (Hh-hpl-), including the knockout strain generated in Chapter 3. During an extended co-culture assay, the com-petitive advantage of Hh-hpl\\(^+\\) strains was evident within two generations, culminating in a total loss of NTHi fitness over subsequent generations. Hh strains capable of high levels of hpl expression, were able to compete with NTHi more effectively, providing a strong link between the NTHi-inhibitory phe-notype, hpl expression and favourable outcomes during competitive growth with NTHi in vitro. These findings demonstrate that Hh-Hpld to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl\\(^+\\) strains possess characteristics desirable in a probiotic, warranting further investigation into the utility of these strains in preventing NTHi interactions with host cells. In chapter 5, the capacity of Hh-Hpl\\(^+\\) strains to disrupt NTHi association with airway epithelial cells was tested to determine their probiotic utility against the requisite nasopharyngeal colonisation stage of NTHi infection. Cell culture models of nasopharyngeal (D562) and lung (A549) epithelia were pre-treated with Hh strains with different levels of hpl expression prior to NTHi challenge. NTHi attach-ment and invasion was significantly reduced in cell monolayers pre-treated with Hpl or Hh strains with high levels of hpl expression. Among all Hh-Hpl\\(^+\\) strains the production of Hpl was found to be stimulated in response to NTHi challenge and nasopharyngeal cell exposure. Pre-treatment with Hh-Hpl\\(^+\\) strains was more effective than the purified Hpl protein alone, such that Hh-Hpl\\(^+\\) cell numbers 10-100-fold lower than that of the NTHi challenge load, were sufficient to significantly inhibit NTHihost-cell interactions. These data suggest that conditions in the nasopharyngeal niche might support high levels of expression of hpl in Hh-Hpl\\(^+\\) strains with associated protection against NTHi adhesion and attachment. Based on the in vitro findings presented in Chapters 3-5, we hypothesised that natural pharyngeal carriage of Hh strains with the Hh-hpl\\(^+\\) genotype would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Chapter 6 describes an in vivo human study involving the collection of oropharyngeal swabs from 257 healthy adults in Australia between 2018 and 2019. 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. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was also assessed in individuals that carried either Hh-hpld to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl\\(^+\\) or Hh-hpl\\(^-\\) (n = 25) or Hh-hpl\\(^+\\) (n = 25). The carriage of Hh-hpl\\(^+\\) was associated with a significantly lower proportionate density and prevalence of concurrent NTHi carriage. Additionally, colonisation with high densities of Hh-hpl\\(^+\\) correlated with lower NTHi carriage loads and a lower likelihood of acquiring/maintaining NTHi colonisation status between visits. These findings suggest a potential pro-tective role of Hh-hpl\\(^+\\) strains against NTHi pharyngeal colonisation in vivo. In summary, the work presented in this thesis provides in vitro and in vivo evidence which supports the therapeutic potential of Hh-Hpl\\(^+\\) against NTHi by inhibiting growth and host-cell interactions re-quired for pathogenesis. These findings encourage translational studies of a probiotic which can be applied to the upper respiratory tract as a strategy to prevent NTHi infections which is not compro-mised by the limitations associated with standard antibiotic or vaccination strategies.