Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.

The Respiratory Microbiome in Paediatric Chronic Wet Cough: What Is Known and Future Directions.

Chronic wet cough for longer than 4 weeks is a hallmark of chronic suppurative lung diseases (CSLD), including protracted bacterial bronchitis (PBB), and bronchiectasis in children. Severe lower respiratory infection early in life is a major risk factor of PBB and paediatric bronchiectasis. In these conditions, failure to clear an underlying endobronchial infection is hypothesised to drive ongoing inflammation and progressive tissue damage that culminates in irreversible bronchiectasis. Historically, the microbiology of paediatric chronic wet cough has been defined by culture-based studies focused on the detection and eradication of specific bacterial pathogens. Various 'omics technologies now allow for a more nuanced investigation of respiratory pathobiology and are enabling development of endotype-based models of care. Recent years have seen substantial advances in defining respiratory endotypes among adults with CSLD; however, less is understood about diseases affecting children. In this review, we explore the current understanding of the airway microbiome among children with chronic wet cough related to the PBB-bronchiectasis diagnostic continuum. We explore concepts emerging from the gut-lung axis and multi-omic studies that are expected to influence PBB and bronchiectasis endotyping efforts. We also consider how our evolving understanding of the airway microbiome is translating to new approaches in chronic wet cough diagnostics and treatments.

Oropharyngeal Carriage of hpl-Containing Haemophilus haemolyticus Predicts Lower Prevalence and Density of NTHi Colonisation in Healthy Adults.

Nontypeable Haemophilus influenzae (NTHi) is a major respiratory pathogen that initiates infection by colonising the upper airways. Strategies that interfere with this interaction may therefore have a clinically significant impact on the ability of NTHi to cause disease. We have previously shown that strains of the commensal bacterium Haemophilus haemolyticus (Hh) that produce a novel haem-binding protein, haemophilin, can prevent NTHi growth and interactions with host cells in vitro. We hypothesized that natural pharyngeal carriage of Hh strains with the hpl open reading frame (Hh-hpl+) would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Oropharyngeal swabs were collected 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 assessed in individuals that carried either Hh-hpl- (n = 25) or Hh-hpl+ (n = 25). 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 or 2.67 times less likely to carry NTHi in their oropharynx, respectively. Colonisation with high densities of Hh-hpl+ correlated with a low NTHi carriage load and a 2.63 times lower likelihood of acquiring/maintaining NTHi colonisation status between visits. Together with supporting in vitro studies, these results encourage further investigation into the potential use of Hh-hpl+ as a respiratory probiotic candidate for the prevention of NTHi infection.

Haemophilin-Producing Strains of Haemophilus haemolyticus Protect Respiratory Epithelia from NTHi Colonisation and Internalisation.

Nontypeable Haemophilus influenzae (NTHi) is a significant respiratory tract pathogen responsible for infections that collectively pose a substantial health and socioeconomic burden. The clinical course of these infections is largely dictated by NTHi interactions with host respiratory epithelia, and thus, approaches that disrupt colonisation and invasion may have significant therapeutic potential. Survival, successful host-cell interactions, and pathogenesis are reliant on NTHi's ability to sequester host-derived haem. Previously, we demonstrated the therapeutic potential of exploiting this haem-dependence using a closely related competitor bacterium, Haemophilus haemolyticus (Hh). Hh strains capable of producing the novel haem-binding protein haemophilin (Hpl) possessed potent inhibitory activity by restricting NTHi access to haem in a broth co-culture environment. Here, we extend this work to cell culture models that more closely represent the human respiratory epithelium and show that Hh strains with high levels of hpl expression protect epithelial cell line monolayers against adhesion and invasion by NTHi. Inhibitory activity was dependent on the level of Hpl production, which was stimulated by NTHi challenge and nasopharyngeal cell exposure. Provided these protective benefits translate to in vivo applications, Hpl-producing Hh may have probiotic utility against NTHi infections by inhibiting requisite nasopharyngeal colonisation.

In Vitro Anti-NTHi Activity of Haemophilin-Producing Strains of Haemophilus haemolyticus.

Nontypeable Haemophilus influenzae (NTHi) is a leading causative organism of opportunistic respiratory tract infections. However, there are currently no effective vaccination strategies, and existing treatments are compromised by antibiotic resistance. We previously characterized Haemophilus haemolyticus (Hh) strains capable of producing haemophilin (HPL), a heme-binding protein that restricts NTHi growth by limiting its access to an essential growth factor, heme. Thus, these strains may have utility as a probiotic therapy against NTHi infection by limiting colonization, migration and subsequent infection in susceptible individuals. Here, we assess the preliminary feasibility of this approach by direct in vitro competition assays between NTHi and Hh strains with varying capacity 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. HPL-producing strains of Hh not only outcompeted NTHi during short-term and extended co-culture, but also demonstrated a growth advantage compared with Hh strains unable to produce the protein. Additionally, HPL expression levels during competition correlated with the NTHi-inhibitory phenotype. HPL-producing strains of Hh demonstrate significant probiotic potential against NTHi colonization in the upper respiratory tract, however, further investigations are warranted to demonstrate a range of other characteristics that would support the eventual development of a probiotic.

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae.

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.

New therapeutic targets for the prevention of infectious acute exacerbations of COPD: role of epithelial adhesion molecules and inflammatory pathways.

Chronic respiratory diseases are among the leading causes of mortality worldwide, with the major contributor, chronic obstructive pulmonary disease (COPD) accounting for approximately 3 million deaths annually. Frequent acute exacerbations (AEs) of COPD (AECOPD) drive clinical and functional decline in COPD and are associated with accelerated loss of lung function, increased mortality, decreased health-related quality of life and significant economic costs. Infections with a small subgroup of pathogens precipitate the majority of AEs and consequently constitute a significant comorbidity in COPD. However, current pharmacological interventions are ineffective in preventing infectious exacerbations and their treatment is compromised by the rapid development of antibiotic resistance. Thus, alternative preventative therapies need to be considered. Pathogen adherence to the pulmonary epithelium through host receptors is the prerequisite step for invasion and subsequent infection of surrounding structures. Thus, disruption of bacterial-host cell interactions with receptor antagonists or modulation of the ensuing inflammatory profile present attractive avenues for therapeutic development. This review explores key mediators of pathogen-host interactions that may offer new therapeutic targets with the potential to prevent viral/bacterial-mediated AECOPD. There are several conceptual and methodological hurdles hampering the development of new therapies that require further research and resolution.