Identification and characterization of the novel CEACAM-binding adhesin of Haemophilus influenzae

Haemophilus influenzae (Hinf), a mostly commensal inhabitant of human respiratory airways, is the causative agent of local infections such as middle ear infections, sinusitis, bronchitis, conjunctivitis and pneumonia, but can also cause life-threatening disseminating diseases, including meningitis and septicemia. The success of Hinf as commensal or opportunistic pathogen relies on the ability of the bacterium to use a combination of adhesive surface proteins, the so-called adhesins, which intimately bind to structures on the human mucosal tissue in order to overcome mechanical clearance and escape immune recognition. In that respect, Hinf targets carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are glycoproteins of the immunoglobulin superfamily and which are present on the apical side of nasopharyngeal epithelial cells. The first chapter of this work presents in detail the numerous advantages for the bacteria inherent in their interaction with epithelial CEACAMs (e.g.: strong attachment to the mucosal surface, internalization within epithelial cells and therefore protection against immune response).The interaction between Hinf and CEACAMs was postulated to be mediated by the outer membrane protein (OMP) P5, one of the major OMPs of Hinf. Therefore, we set out to characterize the molecular requirements of CEACAM-binding by OMP P5. However, (i) the ability of P5-deficient Hinf to strongly interact with CEACAMs; (ii) the similarity of P5 sequences from non-CEACAM-binding and CEACAM-binding Hinf strains, and (iii) the failure of OMP P5 to bind CEACAMs when heterologously expressed in Escherichia coli questioned the role of OMP P5 as the Hinf CEACAM-binding adhesin. A screen of a panel of Hinf mutants with defined deletions in their outer membrane proteins revealed that the depletion of OMP P1 totally abrogated the interaction of Hinf with CEACAMs. Furthermore, non-CEACAM-binding E. coli gained CEACAM-binding properties upon ectopic expression of Hinf OMP P1. Thus, OMP P1 is necessary and sufficient for CEACAM-targeting.Following the identification of OMP P1 as the bona fide CEACAM-binding adhesin of Hinf, we sought to identify the domain(s) of OMP P1 involved in CEACAM-recognition. Therefore, we took advantage of the high similarity between OMP P1 and its non-CEACAM-binding homolog FadL in E. coli. The solved crystal structure of FadL helped to model OMP P1, which is predicted to build a 14-stranded β-barrel. Interestingly, chimeras resulting from the fusion of OMP P1 with FadL fragments combined with OMP P1 mutants deprived of specific extracellular loops clearly underlined the involvement of the most prominent surface-exposed OMP P1 loops (L1, L3, L4 and L7) in CEACAM recognition. The flexibility of these loops appears important for the proper binding to CEACAM. In contrast to Hinf OMP P1, P1 homologs of other members of the Pasteurellaceae family were unable to recognize CEACAMs. Moreover, Hinf OMP P1 bound exclusively to human CEACAMs, but not to other distantly related mammalian CEACAMs.FadL, the OMP P1 homolog in E. coli, mediates the transport of long-chain fatty acids (LCFAs) across the bacterial outer membrane. The high structural homology shared by FadL and OMP P1 raised the question whether OMP P1 is involved in fatty acid transport and/or is regulated by fatty acids. Though the addition of fatty acids to the Hinf medium was able to potently stimulate bacterial growth, the role of OMP P1 in LCFA transport requires further investigations. However, it was obvious that LCFAs supplementations upregulated OMP P1 expression in Hinf. Furthermore, Hinf strains treated with LCFAs were able to better interact with recombinant CEACAMs and were more invasive in CEACAM-expressing epithelial cells, compared to untreated bacteria.In sum, this study not only identifies a novel CEACAM-binding adhesin in a prominent human pathogen, but also provides insights into a new CEACAM-binding interface built by 4 flexible loops of a bacterial outer membrane protein.publishe