Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

Effect of LOS truncation on NTHi 375 biofilm formation under continuous-flow conditions, in microfermentors containing the glass slides where bacteria formed the biofilm. Images are from a representative experiment for wild-type NTHi 375 and Δlic1, Δlic1ABCD-c, ΔsiaB, and ΔopsX mutants. The bottom panel shows the OD₆₀₀ of bacterial suspension detached from glass slide for each strain. Data shown are means and SDs of absorbance values, compared by a two-tailed t test, versus those obtained for NTHi 375. ^aP < 0.005; ^bP < 0.0005.

Infection of A549 epithelial cells by NTHi strains with serial LOS truncation. (A) Effect of LOS truncation on NTHi epithelial adhesion. Wild-type NTHi 375 and Δlic1, ΔsiaB, Δlic2A, ΔlgtF, ΔlpsA, ΔlgtF ΔlpsA, and ΔopsX mutants were used to infect cells for 30 min. The number of bacteria adhered/well is shown for each strain. Mean numbers were significantly lower (*, P < 0.05) than those obtained for the wild-type strain for the ΔlgtF, ΔlgtF ΔlpsA, and ΔopsX mutants. (B) Effect of LOS truncation on NTHi epithelial cell invasion. Wild-type NTHi 375 and Δlic1, ΔsiaB, Δlic2A, ΔlgtF, ΔlgtF-c, ΔlpsA, ΔlgtF ΔlpsA, and ΔopsX mutants were used to infect cells for 2 h. The number of invading bacteria/well is shown for each strain. Mean numbers were significantly lower (*, P < 0.0001) than those obtained for the wild-type strain for the ΔlgtF, ΔlgtF ΔlpsA, and ΔopsX mutants. (C) Effect of LOS truncation on NTHi-mediated inflammatory response. IL-8 secreted to the supernatant by cells upon infection with wild-type NTHi 375 and the Δlic1, Δlic2A, ΔsiaB, ΔlgtF, ΔlpsA, ΔlgtF ΔlpsA, and ΔopsX mutants was quantified at 6 (white bars) and 20 (gray bars) h postinfection by ELISA. CON, noninfected cells. Mean numbers for the ΔopsX mutant were significantly (*, P < 0.001) higher than those obtained for the wild-type strain at 6 and 20 h postinfection.

Histopathological findings and lesion scores in the respiratory system of CD1 mice intranasally infected with NTHi 375. (Top) Tissue sections were H&E stained and lesions subjectively scored on a scale from 0 to 3 (0, absent; 1, mild; 2, moderate; and 3, severe) for inflammatory markers. Selected tissue sections were also IHC stained to detect NTHi 375 antigen in the lesions. The left column shows upper airways from an infected mouse analyzed at 24 h p.i. (U1) Mild epithelial degeneration and necrosis and small amounts of luminal exudate (H&E). (U2) Detail of the lumen of the turbinate filled with serous exudate, neutrophils, and detached epithelial cells (H&E). (U3) NTHi antigen located in the cytoplasm of neutrophils in the luminal exudate (IHC). The middle column shows the trachea from an infected mouse sacrificed at 48 h p.i. (T1) Seropurulent exudate in the tracheal lumen (H&E). (T2) Detail of the exudate from T1 (H&E). (T3) Immunostained NTHi antigen located in the cytoplasm of large phagocytic cells present in the luminal exudate (IHC). The right column shows a lung from an infected mouse analyzed at 48 h p.i. (L1) Large areas of acute bronchopneumonia. (L2) Detail of alveolar spaces and thickened alveolar septa filled with neutrophils and macrophages. (L3) NTHi antigen in the cytoplasm of some phagocytic cells (IHC). (Bottom) Histopathological average lesion scores obtained from sections of upper airways (left), tracheas (middle), and lungs (right) at 24, 48, and 72 h p.i. In the upper airways, the most severe lesions were observed at 24 h p.i., whereas in trachea and lungs, the lesions peaked at 48 h p.i.

Bacterial loads in the lungs of CD1 mice infected by NTHi strains with serial LOS truncation. Mice were infected intranasally with ∼10⁸ bacteria. Bacterial counts in lungs at 24 or 48 h p.i. were determined. Results are reported as log₁₀ CFU/lung and represented as box plot graphs (lines inside boxes represent median values). Statistical differences versus the wild-type strain are indicated as follows: a, P < 0.05; b, P < 0.01; and c, P < 0.001.