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Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways.

Euba B, Moleres J, Viadas C, Ruiz de los Mozos I, Valle J, Bengoechea JA, Garmendia J - PLoS ONE (2015)

Bottom Line: Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20.We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant.Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain.

ABSTRACT
Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

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Analysis of P5 and Hap involvement in NTHi375 interaction with host cell receptors.(A) P5 from NTHi375 is not a ligand for CEACAM1. Effect of the ompP5 and hap gene disruption on NTHi epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. (B) P5 from Hi RdKW20 is a ligand for CEACAM1. Effect of the ompP5 gene disruption on Hi RdKW20 epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. P5 and Hap are not involved in NTHi375 interplay with α5 integrin and N-glycosylated host cell molecules. A549 cells were left untreated (CON) or were pre-incubated with blocking anti-α5 P1D6 antibody, RGD peptide, or tunicamycin, and NTHi375 (C), ΔompP5(D), Δhap(E) and ΔompP5Δhap(F) bacterial adhesion was determined. Data are shown as % adhesion related to control untreated cells. Experiments were performed in triplicate in at least three independent occasions (n≥9).
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pone.0123154.g003: Analysis of P5 and Hap involvement in NTHi375 interaction with host cell receptors.(A) P5 from NTHi375 is not a ligand for CEACAM1. Effect of the ompP5 and hap gene disruption on NTHi epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. (B) P5 from Hi RdKW20 is a ligand for CEACAM1. Effect of the ompP5 gene disruption on Hi RdKW20 epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. P5 and Hap are not involved in NTHi375 interplay with α5 integrin and N-glycosylated host cell molecules. A549 cells were left untreated (CON) or were pre-incubated with blocking anti-α5 P1D6 antibody, RGD peptide, or tunicamycin, and NTHi375 (C), ΔompP5(D), Δhap(E) and ΔompP5Δhap(F) bacterial adhesion was determined. Data are shown as % adhesion related to control untreated cells. Experiments were performed in triplicate in at least three independent occasions (n≥9).

Mentions: It has been previously reported that, depending on the strain, P5 could be a bacterial ligand for the eukaryotic receptor CEACAM1 [14]. NTHi375 adhesion assays were performed by using HeLa-BGP (biliary glycoprotein or CD66a, currently known as CEACAM1) cells [34], a HeLa derivative cell line stably expressing hCEACAM1-4L [5], and previously used to assess the impact of CEACAM1 on bacterial infections [5,39]. NTHi375 showed similar adhesion to HeLa control and HeLa-BGP cells, excluding a potential role for CEACAM1 in NTHi375 epithelial adhesion. Adhesion of NTHi375ΔompP5 and ΔompP5Δhap was lower than that displayed by the wild-type strain, to the same extent for both for HeLa (p<0.0001 and p<0.0005, respectively) and HeLa-BGP (p<0.0005) cells (Fig 3A). These results suggest that NTHi375 does not interact with CEACAM1, that P5NTHi375 is not likely to be a ligand for CEACAM1, and that P5, but not Hap, participates in NTHi375 adhesion to HeLa epithelial cells.


Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways.

Euba B, Moleres J, Viadas C, Ruiz de los Mozos I, Valle J, Bengoechea JA, Garmendia J - PLoS ONE (2015)

Analysis of P5 and Hap involvement in NTHi375 interaction with host cell receptors.(A) P5 from NTHi375 is not a ligand for CEACAM1. Effect of the ompP5 and hap gene disruption on NTHi epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. (B) P5 from Hi RdKW20 is a ligand for CEACAM1. Effect of the ompP5 gene disruption on Hi RdKW20 epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. P5 and Hap are not involved in NTHi375 interplay with α5 integrin and N-glycosylated host cell molecules. A549 cells were left untreated (CON) or were pre-incubated with blocking anti-α5 P1D6 antibody, RGD peptide, or tunicamycin, and NTHi375 (C), ΔompP5(D), Δhap(E) and ΔompP5Δhap(F) bacterial adhesion was determined. Data are shown as % adhesion related to control untreated cells. Experiments were performed in triplicate in at least three independent occasions (n≥9).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403991&req=5

pone.0123154.g003: Analysis of P5 and Hap involvement in NTHi375 interaction with host cell receptors.(A) P5 from NTHi375 is not a ligand for CEACAM1. Effect of the ompP5 and hap gene disruption on NTHi epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. (B) P5 from Hi RdKW20 is a ligand for CEACAM1. Effect of the ompP5 gene disruption on Hi RdKW20 epithelial adhesion to CEACAM1, assessed by infection of HeLa-BGP and HeLa control epithelial cells. P5 and Hap are not involved in NTHi375 interplay with α5 integrin and N-glycosylated host cell molecules. A549 cells were left untreated (CON) or were pre-incubated with blocking anti-α5 P1D6 antibody, RGD peptide, or tunicamycin, and NTHi375 (C), ΔompP5(D), Δhap(E) and ΔompP5Δhap(F) bacterial adhesion was determined. Data are shown as % adhesion related to control untreated cells. Experiments were performed in triplicate in at least three independent occasions (n≥9).
Mentions: It has been previously reported that, depending on the strain, P5 could be a bacterial ligand for the eukaryotic receptor CEACAM1 [14]. NTHi375 adhesion assays were performed by using HeLa-BGP (biliary glycoprotein or CD66a, currently known as CEACAM1) cells [34], a HeLa derivative cell line stably expressing hCEACAM1-4L [5], and previously used to assess the impact of CEACAM1 on bacterial infections [5,39]. NTHi375 showed similar adhesion to HeLa control and HeLa-BGP cells, excluding a potential role for CEACAM1 in NTHi375 epithelial adhesion. Adhesion of NTHi375ΔompP5 and ΔompP5Δhap was lower than that displayed by the wild-type strain, to the same extent for both for HeLa (p<0.0001 and p<0.0005, respectively) and HeLa-BGP (p<0.0005) cells (Fig 3A). These results suggest that NTHi375 does not interact with CEACAM1, that P5NTHi375 is not likely to be a ligand for CEACAM1, and that P5, but not Hap, participates in NTHi375 adhesion to HeLa epithelial cells.

Bottom Line: Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20.We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant.Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain.

ABSTRACT
Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Show MeSH
Related in: MedlinePlus