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

Abstract

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.

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

“…To investigate whether hpl expression is altered following exposure of Hh strains to mammalian cultured cells or NTHi, we performed an analysis of hpl mRNA and Hpl protein levels following co-culture experiments. Baseline hpl expression in cell culture media was highest in Hh-BW1 and Hh-RHH122, approximately 10-fold lower in Hh-NF5 ( Figure 4 A), and absent in Hh-NF4 and Hh-NF1 (data not shown), which is consistent with findings in bacterial growth medium [ 40 ]. Hpl mRNA and protein levels did not differ significantly between RPMI and MEM cell culture media ( Figure 4 A,B, p < 0.05).…”

“…We previously demonstrated the NTHi-inhibitory capacity of Hpl-producing Hh strains in a broth co-culture environment and proposed the probiotic utility of these strains against NTHi colonisation of the upper respiratory tract [ 40 ]. In the current study, we have extended this work to cell line culture models that more closely represent the human respiratory epithelium and show that some Hh strains with high levels of hpl expression protect epithelial cell line monolayers against adhesion and invasion by NTHi.…”

“…In this study, we used five isolates of Hh with differing abilities to inhibit the growth of NTHi in broth co-cultures [ 40 ]. In these strains, the level of hpl expression correlates with the ability of the strain to inhibit adhesion and invasion of model epithelial cell lines by NTHi.…”

“…We have previously shown that Hpl exhibits NTHi growth-inhibitory activity owing to its capacity to bind haem in a form that is inaccessible to NTHi [ 39 ]. Although the mechanism by which Hpl acts to protect model epithelial cell lines against invasion by NTHi is not explored in this study, it seems likely that this is also a result of limited haem availability to NTHi, resulting in growth inhibition [ 39 , 40 ]. NTHi lacks the necessary enzymes required for de novo haem synthesis, and the deleterious effects of haem starvation on NTHi are demonstrated by NTHi strains lacking either the HxuCBA, PE, SapABCDFZ, or HbpA-DppBCDF haem-acquisition systems.…”

“…Previously, we have shown that a closely related bacterium, Haemophilus haemolyticus (Hh), competes with NTHi for limited haem resources. Hh strains capable of producing the novel haem-binding protein haemophilin (Hpl) possessed potent inhibitory activity by restricting NTHi access to haem in vitro [ 38 , 39 , 40 ]. These findings suggest that Hh strains capable of producing high levels of Hpl might disrupt NTHi association with airway epithelial cells and have potential as a probiotic that prevents the requisite nasopharyngeal colonisation stage of infection.…”