Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic ON ). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic ON . The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.Nontypeable Haemophilus influenzae (NTHI) is a commensal of the human upper airways that can cause localized opportunistic airway infections when mucociliary defenses are compromised (24). NTHI is a leading cause of otitis media with effusion (2), acute otitis media (1), chronic sinusitis (20), and pulmonary infections associated with chronic obstructive pulmonary disease (31). For many of these infections, NTHI bacteria persist within dense biofilm communities that are thought to provide resistance to host clearance (12,25,29). NTHI biofilms contain variants expressing lipooligosaccharides (LOS) that contain phosphorylcholine (PCho) and sialic acid (NeuAc) (10,19,36,41). Mutants lacking these structures are unable to establish persistent biofilms in vivo (14,19,36). The addition of PCho to LOS occurs in a phase-variable manner, and thus, NTHI populations contain discrete subpopulations of PCho-positive (PCho ϩ ) and PCho-negative (PCho Ϫ ) variants (40). PCho contributes to NTHI colonization and persistence by promoting bacterial adherence to host cells (9,32,...

Nontypeable Haemophilus influenzae (NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation, PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated with PCho substitutions. We thus conclude that biofilm growth increases the proportion of PCho ؉ variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.Nontypeable Haemophilus influenzae (NTHI) is a humanspecific respiratory commensal that is especially common in young children (12) and patients with impaired mucociliary clearance (36). In contrast with encapsulated H. influenzae strains that cause acute invasive infections, NTHI causes localized airway infections (36). These infections include otitis media (4, 47), bronchitis (45), and sinusitis (22), and are among the most common and costly public health problems worldwide.Most of the NTHI "virulence factors" identified to date are determinants of asymptomatic carriage that allow the organism to persist in the airways. These include adherence factors (48), immunoglobulin A protease (55), and endotoxin (50). Like many bacteria adapted to mucosal surfaces, NTHI endotoxins are lipooligosaccharides (LOS) which lack polymeric O side chains (43,51). Innate responses to the lipid A portion of LOS are mediated by Toll-like receptor 4 (TLR4) (39) and are important to the containment of H. influenzae infections in the airway (56) and in the generation of the inflammatory response that is the hallmark of chronic airway infections (10). Our previous work showed that production of the hexa-acylated lipid A recognized by TLR4 promotes persistent NTHI colonization (50), at least in part by increasing bacterial resistance to killing by defensins (46).The carbohydrate portion of NTHI LOS is highly variable (3...

The airways of patients with chronic obstructive pulmonary disease (COPD) are continually colonized with bacterial opportunists like nontypeable Haemophilus influenzae (NTHi), and a wealth of evidence indicates that changes in bacterial populations within the lung can influence the severity of COPD. In this study, we used a murine model for COPD/emphysema to test the hypothesis that COPD affects pulmonary clearance. Mice were treated with a pulmonary bolus of elastase, and as reported previously, the lungs of these mice were pathologically similar to those with COPD/emphysema at ϳ1 month posttreatment. Pulmonary clearance of NTHi was significantly impaired in elastase-treated versus mock-treated mice. While histopathologic analysis revealed minimal differences in localized lung inflammation between the two groups, lower levels of intercellular adhesion molecule 1 (ICAM-1) were observed for the airway epithelial surface of elastase-treated mice than for those of control mice. Following infection, elastase-treated mice had lung pathology consistent with pneumonia for as long as 72 h postinfection, whereas at the same time point, mock-treated mice had cleared NTHi and showed little apparent pathology. Large aggregates of bacteria were observed within damaged lung tissue of the elastase-treated mice, whereas sparse individual bacteria were observed in lungs of mock-treated mice at the same time point postinfection. Additional infection studies showed that NTHi mutants with biofilm defects were less persistent in the elastase-treated mice than the parent strain. These findings establish a model for COPD-related infections and support the hypotheses that ICAM-1 promotes clearance of NTHi. Furthermore, the data indicate that NTHi may form biofilms within the context of COPD-related infections.

؊/؊ mice. These results indicate that PCho promotes bacterial resistance to pulmonary clearance early in infection in a manner that is at least partially independent of the TLR4 pathway.